Escherichia coli CMP-NeuAc synthetase catalyses formation of cytidine 5' monophospho-N-acetylneuraminic acid (CMP-NeuAc) from N-acetylneuraminic acid (NeuAc) and cytidine triphosphate (CTP). Availability and specificity of purified sugar activating enzymes and glycosyltransferases make these enzymes convenient tools for synthesis of biologically relevant oligosaccharides. The role of the cysteines in the structural and catalytic properties of the enzyme has been examined by site directed mutagenesis and chemical modification. Chemical modification with the sulfhydro specific reagent DTDP suggests that one cysteine residue is involved in catalysis, since that enzyme can be completely inactivated by titration of one cysteine residue with DTDP. The enzyme can be protected from inactivation in the presence of the substrate CTP. Site directed mutagenesis demonstrates that cysteine 129 and cysteine 329 are not essential for catalysis, since both can be substituted by selected amino acids without complete loss of activity. Chemical inactivation with N- ethylmaleimide (NEM) demonstrates that cysteine 329 and cysteine 129 are inaccessible to NEM at room temperature. Cysteine 329 can be thermally exposed to chemical modification with NEM at 42 degree C but cysteine 129 is inaccessible to chemical modification with NEM. We have sequenced the amino terminus of the CMP NeuAc synthetase from meningococcus group B which synthesizes the same a 2-8 NeuAc capsule of E. coli K 1. The N terminal sequences were compared and a concensus obtained. Arginine and lysine residues were identified as common amino acids in the compared sequences. We have mutated 14 arginines in E. coli CMP NeuAc synthetase to Glycine and mutation of the common arginine completely inactivates CMP NeuAc synthetase. Changing arginine to alanine results in an inactivated enzyme, but a change to lysine restores the specific activity to wild type values. Km determinations are being done at different pHs to determine involvement of arginine in catalysis.